Biotechnology for Biofuels最新文献

Background

2,3-butanediol (2,3-BDO) is an economically important platform chemical that can be produced by the fermentation of sugars using an engineered strain of Zymomonas mobilis. These fermentations require continuous monitoring and modification of fermentation conditions to maximize 2,3-BDO yields and minimize the production of the undesired coproducts glycerol and acetoin. Because of the time required for sampling and off-line chromatographic measurement of fermentation samples, the ability of fermentation scientists to modify fermentation conditions in a timely manner is limited. The goal of this study was to test if near-infrared spectroscopy (NIRS) along with multivariate statistics could reduce the time needed for this analysis and enable real-time monitoring and control of the fermentation.

Results

In this work we developed partial least squares (PLS) calibration models to predict the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol in fermentations via NIRS using two different spectrometers and two different spectroscopy modalities. We first evaluated the feasibility of rapid NIRS monitoring through experiments where we measured the signals from each analyte of interest and built NIRS-based PLS models using spectra from synthetic samples containing uncorrelated concentrations of these analytes. All analytes showed unique spectral signatures, and this initial modeling showed that all analytes could be detected simultaneously. We then began work with samples from laboratory fermentation experiments and tested the feasibility of regression model development across two spectral collection modalities (at-line and on-line) and two instruments: a laboratory-grade instrument and a low-cost instrument with a more limited spectral range. All modalities showed promise in the ability to monitor Z. mobilis fermentations of glucose and xylose to 2,3-BDO. The low-cost instrument displayed a lower signal-to-noise ratio than the laboratory-grade instrument, which led to comparatively lower performance overall, but still provided sufficient accuracy to monitor fermentation trends. While the ease of use of on-line monitoring systems was favored as compared to at-line systems due to the lack of sampling required and potential for automated process control, we observed some decrease in performance due to the additional complexity of the sample matrix.

Conclusion

We have demonstrated that NIRS combined with multivariate analysis can be used for at-line and on-line monitoring of the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol during Z. mobilis fermentations. The decrease in signal-to-noise ratio when using a low-cost spectrometer led to greater prediction error than the laboratory-grade spectrometer for at-line monitoring. The on-line monitoring modality showed great promise for real time process control via NIRS.

Background

Large-scale cultivation of microalgae provides a carbon–neutral source of biomass for extracting valuable compounds and producing renewable fuels. Owing to their high metabolic activity and rapid reproduction rates, Chlorella species are highly productive when grown in photobioreactors. However, wild-type strains have some biological limitations that make algal bioproducts more expensive than those from more traditional sources. Domestication is thus required for improving strains. Engineering Chlorella species has been made difficult by their chemically complex and highly resistant cell wall, making transformation difficult. Cell wall also restricts diffusion of organic solvents; thus, limiting the extraction of valuable intracellular compounds. Obtaining strains with weakened cell wall is crucial to enhance the extractability of intracellular molecules, reducing the costs of biomass disruption, and to improve genetic transformation efficiency.

Results

We developed a mutagenesis pipeline combined with single-cell fluorescence scanning on the microalga Chlorella vulgaris to identify mutants with altered cell wall properties. We used the fluorescent dyes erythrosin B and calcofluor white, as markers for cell wall permeability and for binding the structural polysaccharides of the cell wall, respectively. Flow cytometry with fluorescence-activated cell sorting was employed to enrich mutagenized populations with altered emission profiles. After a first round of mutagenesis, we found six mutants with significantly higher cell permeability to erythrosin B than the wild type (CWP lines) and altered cell wall structure and composition. A second round of mutagenesis on a selected CWP strain, followed by selection for lower calcofluor white signal, resulted in the isolation of CFW lines, which exhibited reduced mechanical resistance when the biomass was subjected to cell disruption procedures. This two-steps procedure allowed us to identify new mutant strains with both an increased cell wall permeability and a reduced mechanical resistance, making a novel step towards Chlorella domestication.

Conclusions

This study demonstrated the feasibility of using mutagenesis and phenotypic selection based on flow cytometry screening to alter the cell wall of C. vulgaris and identify promising strains with improved traits for industrial applications.

Bioenergy sorghum is a highly productive drought tolerant C4 grass that accumulates ~ 80% of its harvested biomass in ~ 4 m long stems comprised of > 40 internodes that develop sequentially during an extended vegetative growth phase. Following elongation of each internode, internode density increases ~ threefold to fourfold primarily due to the accumulation of cell walls composed of cellulose, glucuronoarabinoxylan and lignin. Lignin accumulates initially on cell walls of sclerenchyma cells surrounding vascular bundles and later on cell walls of the stem rind and stem core pith parenchyma. Many genes involved in cell wall biosynthesis were expressed continuously during the stem internode densification process whereas others showed dynamic patterns of expression (high to low, low to high). Several CESA genes involved in primary cell wall cellulose synthesis were expressed in the stem rind and core throughout the stem densification phase. In contrast, CESA genes involved in secondary cell wall biogenesis were expressed continuously in the stem rind but downregulated in the stem core shortly after completion of internode elongation. Overall, accumulation of cell wall biomass in elongated internodes during stem densification increases stem mechanical strength and biomass bulk density while modifying biomass composition in ways that could impact the amount and release of cellulosic sugars and lignin-derived bioproducts.

Fossil fuel combustion is a major contributor to the greenhouse effect, which drives global environmental challenges such as climate change. The rapid depletion of fossil fuel reserves necessitates the urgent management of greenhouse gas emissions and the development of sustainable alternatives. Green algae are a promising resource for biofuel production because of their high lipid content (up to 70% dry weight), which can be converted into biofuel. This study investigated the lipid production potential of Tetraselmis sp. under different nutrient media conditions to determine the glucose concentration that maximizes lipid accumulation to advance biofuel research. To determine the effect of glucose concentration on lipid accumulation, Tetraselmis sp. was cultured in three different nutrient media: standard microalgal culture medium (F/2), seawater, and nitrogen-deficient medium (NDM) supplemented with different glucose concentrations. The glucose concentration that maximized lipid accumulation was incorporated into NDM (NDM+G) and effect of the medium was compared with the effects of other media over 9 days. Additionally, reactive oxygen species (ROS) levels and apoptosis rates were measured to assess the cellular effects of glucose supplementation and nitrogen deprivation. NDM+G, with 2 mg/mL glucose, was the most effective medium for lipid accumulation in Tetraselmis sp., with lipid levels peaking significantly (p < 0.05) at 79.8% on day 6 post-glucose supplementation. This suggests that maximum lipid yield can be achieved by harvesting Tetraselmis sp. cultured in glucose-supplemented NDM on day 6. However, ROS levels were elevated significantly (p < 0.05) by day 4, and apoptosis rate reached 31% by day 9, indicating potential cellular stress under the conditions. The use of seawater and cost-effective nutrient formulations improves the industrial feasibility of the approach, while the high lipid yield within a short cultivation period supports its potential application in sustainable large-scale biofuel production. Further research is required to optimize culture conditions using low-cost nitrogen and carbon sources. Such optimization should aim to reduce costs and cellular damage while maximizing lipid production, ultimately enabling more sustainable biofuel solutions.

Fungi play a pivotal role in ecosystem functionality, driving processes such as decomposition, nutrient cycling, and symbiotic interactions. Their wide enzymatic strategies enable the breakdown of complex organic materials and the valorization of organic waste streams, providing sustainable pathways for bioproduct development. Fungi also exhibit significant potential in industrial applications, particularly in biofuel and nutraceutical production, owing to their high lipid content and adaptability to diverse feedstocks. Genera such as Aspergillus, Mortierella, and Linnemannia have demonstrated exceptional lipid production capabilities and unique fatty acid profiles, including high yields of nutraceuticals like arachidonic acid (ARA) and oleic acid. This study explored uncharacterized fungal strains isolated from California grassland soils, analyzing their phylogeny, morphology, growth rates, lipid content, and fatty acid profiles. Results revealed notable genetic and physiological diversity among the isolates, with Mortierella strains emerging as the most promising for industrial applications due to their superior lipid content and productivity of ARA and oleic acid. Confocal microscopy confirmed consistent lipid droplet morphology, while phylogenetic analysis uncovered novel species-level diversity. Key strains were identified for biofuel and nutraceutical production, highlighting their industrial potential. These findings underscore the versatility of fungi as biotechnological tools and provide a foundation for further exploration and utilization of these promising strains in industrial processes.

Graphical Abstract

To propose a strategy for the commercial cultivation of a Korean strain of Nannochloropsis oceanica, the growth, fatty acid content and bacterial community of N. oceanica cultures exposed to different light sources were investigated. Significant growth of N. oceanica cultured under blue (450 nm), red (620 nm) and white (cool-white fluorescent; control) light was observed, whereas growth with relatively low densities was observed in N. oceanica cultured under purple (415 nm) and yellow (592 nm) light. Cells cultured under white and blue light began growing again at day 26, after experiencing stationary phases for 7 days, indicating that day 26 may be a switching point for the growth trajectory in batch culture of N. oceanica. White light also produced the highest biomass of N. oceanica, followed by blue, red, and yellow light. These results indicate that blue and red light, excluding the white light characterized by a wide spectral band, can ensure a high growth rate and biomass of a Korean strain of N. oceanica. With respect to fatty acid content, eicosapentaenoic acid (EPA) was the most dominant under the yellow and red light with N. oceanica exhibiting relatively low biomass dry weight and growth rates. In bacterial communities in N. oceanica cultures exposed to different light sources, the genus Roseovarius appeared to promote the growth of N. oceanica. Based on the results of this study, the most advantageous EPA production system for a Korean strain of N. oceanica initially uses white or blue light to produce the desired cell concentration and rapid growth, then switches to red or yellow light to enhance EPA content. This two-phase cultivation approach offers a viable pathway for large-scale EPA production from native strains, with potential application in nutraceutical or aquaculture industries.

Graphical Abstract

Background

Biohydrogen production from agricultural waste is a promising strategy to address climate change and energy challenges. This study aimed to optimize the process parameters for biohydrogen production from watermelon peels (WMP) by Clostridium butyricum NE133 using statistical optimization techniques. Initial screening of eight significant variables influencing hydrogen production including, initial pH, incubation temperature, WMP concentration, inoculum volume, yeast extract, tryptone, sodium acetate, and ammonium acetate concentration was conducted by a Plackett–Burman (PB) design.

Results

The results showed that four variables including, initial pH (P < 0.001), WMP concentration (P < 0.001), sodium acetate (P = 0.023), and ammonium acetate (P = 0.048) had statistically significant effects on hydrogen production. The model curvature (P = 0.040) indicated that it was significant. Box–Behnken (BB) design under response surface methodology (RSM) was employed to optimize the four selected variables to maximize hydrogen production. The optimal conditions for maximizing hydrogen production from WMP by C. butyricum were: initial pH of 8.98, WMP concentration of 44.75%, sodium acetate 4.49 gL⁻¹, and ammonium acetate 1.15 gL⁻¹ at with predicted H_max of 4703.23 mLL⁻¹. The determination coefficient R² of the model was 0.9902 with the lack of fit F-value was 1.86.

Conclusions

The confirmation experiment revealed only a 0.59% difference between the predicted and experimental hydrogen production, indicating that the optimum conditions were actual with the least error. Improvement of about 103.25% in hydrogen production from WMP by C. butyricum NE133 was achieved after the optimization process.

Graphical Abstract

Paddy straw (PS), a by-product of rice production, has a large volume, low economic value, and environmental impact due to burning, contributing to pollution and health hazards. This manuscript highlights the combined effect of acid treatments and enzymatic hydrolysis of paddy straw to produce fermentable hydrolysate, a potential biofuel. This study uses response surface methodology (RSM) with a Box–Behnken design to optimize process parameters (acid concentration, temperature, and duration of hydrolysis), thereby improving the efficiency of converting paddy straw into fermentable sugars. The efficacy of pretreatment was evaluated based on cellulose content and lignin reduction. The optimal conditions of 1% H₂SO₄, 80 °C, and 20 min resulted in effective cellulose enrichment (95.4%) and lignin reduction (38.2%), promoting efficient enzymatic hydrolysis. The enzymatic hydrolysis used cellulase from Trichoderma reesei, yielding high glucose concentrations of 225.2 mg glucose ml⁻¹ g⁻¹ paddy straw. Using Brunauer–Emmett–Teller (BET) analysis and morphology of pretreated and raw PS samples, the surface modification was validated for the optimized hydrolysis conditions. Surface area and pore volume for optimized condition decreased by 58.6% and 25% respectively. However, mean pore diameter increased by 87.9%. Herein, this study offers a more efficient, optimized, and sustainable pathway for converting paddy straw into biofuel using cellulase, with broader implications for agricultural waste management and renewable energy production.

Graphical Abstract

Background

Microbial lipid extraction is a critical process in the production of biofuels and other valuable chemicals from oleaginous microorganisms. The process involves the separation of lipids from microbial cells. Given the complexity of microbial cell walls and the demand for efficient and environmentally friendly extraction methods, further research is still needed in this area. This study aims to pursue the extraction of intracellular lipids from oleaginous yeasts using inexpensive solvents, without disrupting the cells and even maintaining a certain level of cell viability.

Results

The study used fresh fermentation broth of Rhodotorula toruloides as the lipid extraction target and employed a binary solvent of methyl tert-butyl ether (MTBE) and n-hexane for lipid extraction. The effects of extraction time and solvent ratio on cell viability, lipid extraction efficiency, and fatty acid composition were analyzed. Conditions that balanced lipid yield and cell survival were selected for lipid extraction.

Specifically, using a binary solvent (with 40% MTBE) to extract an equal volume of R. toruloides fermentation broth achieved a total lipid extraction rate of 60%, while maintaining a 5% cell survival rate (the surviving cells served as the seed for the second round of lipid production). After separating the solvent phase and supplementing the lipid-extracted cells with carbon sources and a small amount of nitrogen sources, the cells gradually regained biomass and produced lipids. Repeating this "gentle" extraction on surviving and regrown cells and adding carbon and nitrogen sources can enable a second round of growth and lipid production in these cells.

Conclusions

This is an interesting finding that may potentially encompass the extraction mechanisms of polar/nonpolar solvents and the phenomenon of yeast autophagy. This method does not require the destruction of the cell wall of oleaginous yeast. The separation after extraction is simple, and both the cells and solvents can be recycled. It provides a possible approach for simultaneous fermentation and lipid extraction.

ABRE BINDING FACTOR 4 (ABF4) is a pivotal regulatory gene in the abscisic acid (ABA) signaling pathway, and changes in its expression levels can modulate the plant's stress resistance. To further explore the specific regulatory mechanisms of alternative splicing (AS) in the ABA signaling pathway and to identify new breakthroughs for breeding high stress-resistant varieties of Brassica napus, we identified 17 homologous genes of ABF4 in the genome. Utilizing bioinformatics techniques, we analyzed their motifs, conserved domains, and cis-acting elements of their promoters. Through transcriptome data from the stress-tolerant dwarf strain ndf2 and its parental line 3529, we uncovered a significantly differentially expressed ABF4 gene, which we named BnABF4L. Subsequently, we analyzed the AS events of BnABF4L under normal growth conditions and different abiotic stresses, as well as the impact of different transcript variants' 5’ untranslated region (5'UTR) on gene translation. BnABF4L undergoes alternative 3' splice site (A3SS) selection to produce three transcripts (V1-V3) with divergent 5'UTRs. While V1 translation is suppressed by upstream ORFs (uORFs), V2/V3 exhibit enhanced translational efficiency. Under stress, ndf2 shifts splicing toward V3, circumventing uORF-mediated repression to upregulate stress-adapted isoforms. We validated the inhibitory effect of upstream open reading frames (uORFs) on protein-coding open reading frame (pORFs) and, based on the collective experimental results, proposed the flexible regulatory mechanism of AS events of BnABF4L in response to stress. Our findings provide new insights for future studies on stress resistance in rapeseed as well as for research on the regulation of alternative splicing mechanisms in the ABA signaling pathway.